The long-term goal of this multiple PI proposal is to develop neuroprotective strategies that involve synthesis and testing of mitochondria-targeted antioxidants in a preclinical mouse model of Parkinson's Disease (PD) that can be subsequently translated to patients with PD. In this proposal, we have combined the expertise and experience of individuals in synthetic organic chemistry, free radical biology, neuropharmacology, and neurotoxicology from two institutions, the Medical College of Wisconsin and Iowa State University. This proposal is based on the discovery that mitochondria- targeted antioxidants (MTAs) inhibit oxidative stress and neuronal damage in 1-methyl-4- phenylpyridinium (MPP+) treated neuronal cell culture models of PD as well as in 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) animal models of PD. We hypothesize that MTAs provide an effective neuroprotective strategy for treatment of PD. As a corollary, we propose that MTAs attenuate mitochondria-derived reactive oxygen and nitrogen species (ROS/RNS), thereby protecting against inactivation of key redox sensors in response to mitochondrial neurotoxin exposure. Specifically, we will: (i) Design and synthesize MTAs and assess their cytoprotection in neuronal cell culture models of PD, (ii) Determine the molecular mechanisms of cytoprotection of MTAs in neuronal cell culture models of PD, (iii) Assess the neuroprotective effects of MTAs in a well-established preclinical MPTP mouse model of PD, (iv) Determine the activation/inactivation of key redox targets in mitochondria in response to MTA treatment in the preclinical MPTP mouse model, and (iv) Investigate the long-term tolerability of MTAs during chronic treatment in the mouse model. We will use several analytical techniques (low-temperature EPR, HPLC-fluorescence and electrochemical detection, HPLC/MS, proteomics) to detect and quantitate ROS/RNS, molecular biological approaches (apoptosis measurements, transcription factor translocation) to define the molecular mechanisms, and neurobehavioral and histopathological analyses to evaluate the neuroprotective effects. Abnormal generation of mitochondrial ROS/RNS in response to environmental toxins has been implicated in the pathogenesis of PD. Numerous antioxidants and iron chelators have been used with partial success in experimental animal models of PD. Emerging literature suggests that antioxidants specifically targeted to mitochondria might serve as promising neuroprotectants for treatment of PD. In this proposal, we will assess the neuroprotective efficacy of several novel MTAs in a cell culture and preclinical mouse model of PD. Systematic characterization of neuroprotective properties and long-term tolerability of novel MTAs in both cell culture and animal models will yield comprehensive preclinical data for the clinical development of efficacious mitochondria-targeted antioxidant therapies for PD.